We examined the influence of insulin on S6 kinase activity in skeletal muscle of 7 insulin-sensitive, 4 insulin-resistant, non-diabetic, and 5 diabetic Pima Indians. S6 kinase activity was assayed in extracts biopsies obtained at 0, 15, 30, 45, 60, and 90 minutes during a hyperinsulinemic (-2,000 U/ml plasma), euglycemic clamp. Basal S6 kinase activity was similar in the groups (-0.5 pmol/min mg protein). In sensitive subjects, S6 kinase activity increased sharply between 15 and 30 minutes of insulin infusion, reaching a maximum (4.6-fold increase) at 45 minutes, before 1 declining. S6 kinase activity also increased (3x) in resistant subjects, but the maximum occurred at 60 minutes, without a rapid increase between 15 and 30 minutes. The timecourse of S6 kinase activation in diabetic subjects was similar to that of resistant subjects. The absence of the early response suggested that insulinresistant subjects may lack a particular early-responding S6 kinase. However, chromatography of extracts on FPLC monoQ revealed two peaks of insulin-stimulated S6 kinase activity that eluded identically in the two groups. The majority of the increased activity occurred in peak 2. Immunoblot analysis revealed that the enzyme responsible for peak 1 activity is antigenically related to the 90 kilodalton S6 kinase and that peak 2 activity, which accounts for the majority of the stimulation of total S6 kinase activity by insulin, is accounted for by an enzyme antigenically related to the 70 kilodalton S6 kinase. The abnormal time course of S6 kinase activation in resistant and diabetic subjects is not explained by the lack of a articular S6 kinase but rather by a lower magnitude and rate of stimulation of both S6 kinase activities. These results suggest that the biochemical lesion responsible for insulin resistance in Pima Indians occurs upstream from the S6 kinases in the pathway of insulin signal transduction.